α-Phenyl-N-t-butylnitrone (PBN) is one of the most widely used spin-trapping compounds for investigating the existence of free radicals in biological systems. Recently, there has been considerable interest in the antioxidant nature of PBN on degenerative diseases, presumably related to oxidative stress. In the present study, the protective effect of PBN on the HepG2 cell line under oxidative stress was investigated. When the HepG2 cells were exposed to oxidant, such as hydrogen peroxide, menadione, or ethanol, the protective role of PBN was manifested as a reduction in trypan blue uptake and a decrease in the endogenous production of oxidants, as measured by the oxidation of 2`,7`-dichlorodihydrofluorescin. The modulation of activity of major antioxidant enzymes, such as superoxide dismutase and catalase, was not significantly dittferent either in the presence or in the absence of PBN. This indicates that PBN acts as a direct scavenger of reactive oxygen species.